CN116732024A - Functional modified magnetic bead, DNA gel recovery kit and preparation method and application thereof - Google Patents
Functional modified magnetic bead, DNA gel recovery kit and preparation method and application thereof Download PDFInfo
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- 239000011324 bead Substances 0.000 title claims abstract description 93
- 238000011084 recovery Methods 0.000 title claims abstract description 43
- 238000002360 preparation method Methods 0.000 title abstract description 19
- 239000002245 particle Substances 0.000 claims abstract description 37
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 36
- NBVXSUQYWXRMNV-UHFFFAOYSA-N fluoromethane Chemical compound FC NBVXSUQYWXRMNV-UHFFFAOYSA-N 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 21
- 230000004048 modification Effects 0.000 claims abstract description 20
- 238000012986 modification Methods 0.000 claims abstract description 20
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 17
- -1 dimethyl octenamide dihydrochloride Chemical compound 0.000 claims abstract description 15
- 125000000524 functional group Chemical group 0.000 claims abstract description 12
- 238000000605 extraction Methods 0.000 claims abstract description 10
- 235000012239 silicon dioxide Nutrition 0.000 claims abstract description 10
- 239000000243 solution Substances 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- 239000006087 Silane Coupling Agent Substances 0.000 claims description 9
- 239000011259 mixed solution Substances 0.000 claims description 9
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 8
- BDWFYHUDXIDTIU-UHFFFAOYSA-N ethanol;propane-1,2,3-triol Chemical compound CCO.OCC(O)CO BDWFYHUDXIDTIU-UHFFFAOYSA-N 0.000 claims description 7
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims description 5
- 235000019270 ammonium chloride Nutrition 0.000 claims description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 239000007822 coupling agent Substances 0.000 claims description 3
- FZHAPNGMFPVSLP-UHFFFAOYSA-N silanamine Chemical compound [SiH3]N FZHAPNGMFPVSLP-UHFFFAOYSA-N 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 2
- 238000000576 coating method Methods 0.000 claims description 2
- 238000009833 condensation Methods 0.000 claims description 2
- 230000005494 condensation Effects 0.000 claims description 2
- 230000018044 dehydration Effects 0.000 claims description 2
- 238000006297 dehydration reaction Methods 0.000 claims description 2
- 150000007523 nucleic acids Chemical class 0.000 abstract description 16
- 108020004707 nucleic acids Proteins 0.000 abstract description 13
- 102000039446 nucleic acids Human genes 0.000 abstract description 13
- 238000001179 sorption measurement Methods 0.000 abstract description 9
- 230000000694 effects Effects 0.000 abstract description 6
- 230000003993 interaction Effects 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 239000003094 microcapsule Substances 0.000 abstract description 2
- 239000004005 microsphere Substances 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 45
- 239000000499 gel Substances 0.000 description 25
- 238000005406 washing Methods 0.000 description 23
- 239000007788 liquid Substances 0.000 description 14
- 239000008367 deionised water Substances 0.000 description 12
- 229910021641 deionized water Inorganic materials 0.000 description 12
- 239000012634 fragment Substances 0.000 description 10
- 238000002844 melting Methods 0.000 description 9
- 230000008018 melting Effects 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 238000003756 stirring Methods 0.000 description 9
- 239000006228 supernatant Substances 0.000 description 9
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 description 8
- 239000012148 binding buffer Substances 0.000 description 7
- 238000002156 mixing Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 239000011734 sodium Substances 0.000 description 5
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 238000009826 distribution Methods 0.000 description 4
- 239000003480 eluent Substances 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical group C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
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- 239000008267 milk Substances 0.000 description 3
- 210000004080 milk Anatomy 0.000 description 3
- 235000013336 milk Nutrition 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- 235000017281 sodium acetate Nutrition 0.000 description 3
- 239000001632 sodium acetate Substances 0.000 description 3
- 239000004094 surface-active agent Substances 0.000 description 3
- 238000003260 vortexing Methods 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 229910008051 Si-OH Inorganic materials 0.000 description 2
- 229910006358 Si—OH Inorganic materials 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
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- 239000007853 buffer solution Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
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- 230000009881 electrostatic interaction Effects 0.000 description 2
- 239000012149 elution buffer Substances 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- SJECZPVISLOESU-UHFFFAOYSA-N 3-trimethoxysilylpropan-1-amine Chemical compound CO[Si](OC)(OC)CCCN SJECZPVISLOESU-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 239000003012 bilayer membrane Substances 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000003973 paint Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- UQMOLLPKNHFRAC-UHFFFAOYSA-N tetrabutyl silicate Chemical compound CCCCO[Si](OCCCC)(OCCCC)OCCCC UQMOLLPKNHFRAC-UHFFFAOYSA-N 0.000 description 1
- LFQCEHFDDXELDD-UHFFFAOYSA-N tetramethyl orthosilicate Chemical compound CO[Si](OC)(OC)OC LFQCEHFDDXELDD-UHFFFAOYSA-N 0.000 description 1
- ZQZCOBSUOFHDEE-UHFFFAOYSA-N tetrapropyl silicate Chemical compound CCCO[Si](OCCC)(OCCC)OCCC ZQZCOBSUOFHDEE-UHFFFAOYSA-N 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
- H01F7/00—Magnets
- H01F7/02—Permanent magnets [PM]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Electromagnetism (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Power Engineering (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Crystallography & Structural Chemistry (AREA)
- Soft Magnetic Materials (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
The invention relates to the technical field of microcapsule or microsphere manufacturing, in particular to a functional modification magnetic bead, a DNA gel recovery kit, a preparation method and application thereof. The functional modified magnetic beads of the invention comprise Fe 3 O 4 Core, enwrapping Fe 3 O 4 A silicon dioxide layer and a functional group modified on the surface of the dioxide layer on the surface of the core; the functional group is fluorocarbon and/or dimethyl octenamide dihydrochloride, and the average particle size of the functional modified magnetic beads is 2-10 mu m. The functional modified magnetic beads of the invention are made of Fe 3 O 4 The method is characterized in that the surface of the magnetic bead is coated with a silicon dioxide layer, the silica layer is connected with a functional modification group through a dioxide layer, the interaction between the functional modification group and nucleic acid is utilized to improve the average recovery rate of the magnetic bead to DNA, the extraction rate is more than 80%, and the magnetic bead has good adsorption effect on nucleic acid fragments within the range of 50 bp-50 kb.
Description
Technical Field
The invention relates to the technical field of microcapsule or microsphere manufacturing, in particular to a functional modification magnetic bead, a DNA gel recovery kit, a preparation method and application thereof.
Background
Nucleic acids, including deoxyribonucleic acid (RNA) and ribonucleic acid (DNA), are found predominantly in the nucleus or capsid of the virus, and extraction and purification thereof are of great importance in biomedical science. For extraction of DNA, the DNA gel recovery method is a method for obtaining DNA fragments with specific sizes, and the recovery effect directly influences the subsequent enzyme digestion linkage, transformation screening, sequencing, PCR amplification, labeling, microinjection and other series of experiments. The current commonly used DNA gel recovery method is a column recovery kit, a glass milk/purified filler gel recovery kit, a dialysis live elution method or a magnetic bead gel recovery method, wherein the column recovery kit is suitable for DNA recovery of conventional fragment level (100 bp-10 kb) and small fragment level (< 100 bp); the glass milk/purified packing gel recovery kit and the dialysis live elution method are suitable for DNA recovery of large fragment level (> 7 kb); the magnetic bead gel recovery method is suitable for recovering DNA with various fragment sizes.
However, the above-mentioned DNA gel recovery methods have some problems, such as a column recovery kit, a glass milk/purified filler gel recovery kit, and a dialysis live elution method, which can only be operated manually, cannot be automatically extracted using a machine, and are limited by the size of DNA fragments; although the magnetic bead recovery method is flexible in operation, the operation can be performed manually or by programming, and the automatic extraction can be performed by a machine, the DNA recovery rate of the current magnetic bead recovery method is only 40-50%, and the subsequent experimental requirements are difficult to meet.
Disclosure of Invention
The invention aims to overcome the defect or defect of low DNA recovery rate of the existing magnetic bead recovery method and provide a functional modified magnetic bead.
The invention also aims to provide a preparation method of the functional modification magnetic beads.
A further object of the present invention is the use of the above-described functionally modified magnetic beads for DNA extraction and/or purification.
It is another object of the present invention to provide a DNA gel recovery kit comprising the above-mentioned functionally modified magnetic beads.
The above object of the present invention is achieved by the following technical solutions:
the invention protects a functional modification magnetic bead, which comprises Fe 3 O 4 Core and coating on Fe 3 O 4 A silicon dioxide layer and a functional group modified on the surface of the dioxide layer on the surface of the core;
the functional group is fluorocarbon and/or dimethyl octenamide dihydrochloride; the average particle diameter of the functional modified magnetic beads is 2-10 mu m. Alternatively, the functionally modified magnetic beads have an average particle size of 6 to 7. Mu.m.
It should be noted that: the Fluorocarbon (FS) refers to fluorocarbon surfactant (Capstone FS-61fluorocarbon surfactant), and the brand and manufacturer of the fluorocarbon surfactant are not limited; the CAS number of the dimethyl octenamide Dihydrochloride (DMS) is 34490-86-3.
The particle size of the functional modification magnetic beads and the type and the modification amount of the functional modification groups have important influence on the average recovery rate of DNA. When the functional group is fluorocarbon, the-Si-OH on the surface of the magnetic bead is utilized + C on radicals and fluorocarbon 6 F x -COO - End-bonded, fluorocarbon C 6 C with Fx end and another fluorocarbon 6 F x End-formed bilayer membrane (Magnetic beads-FS-FS), the outermost layer of the modified Magnetic bead is fluorocarbon-COO - At the end of Na + Nucleic acid-OH under concentration regulation + end-COO-capable of being connected with Magnetic beads-FS-FS - The end combination achieves the effect of adsorbing nucleic acid; at the same time, due to Na after washing buffer treatment + Rapidly decreasing Magnetic beads-FS-FS-COO - With nucleic acid-OH + Electrostatic interactions ratio between Magnetic beads-Si-OH + and-COO-FS-FS - The electrostatic interactions between nucleic acids are much weaker, which results in the presence of-COO - Under the dual action of the inversion and elution buffer (pH about 8.5), the ionic bridge linking the nucleic acid may be completely broken through the nucleic acid-OH + Binding to Magnetic beads-FS-FS-COO - The nucleic acid of the group will be released more efficiently and thoroughly, thereby increasing the average recovery of DNA.
When the functional modification group is dimethyl octenamide dihydrochloride, the functional modification group is wrapped in Fe 3 O 4 NH on the surface of the silica layer on the surface of the core 2 + The groups being covalently bound to the DMS such that there is a-NH-at each end of the DMS 2 + A radical, the remaining one NH 2 + The group can be associated with a phosphate group (-PO) in the nucleic acid 4 - ) Binding to achieve the effect of adsorbing nucleic acidThereby improving the average recovery rate of DNA.
In a specific embodiment, the Fe 3 O 4 The average particle diameter of the core is less than or equal to 2 mu m. Fe (Fe) 3 O 4 The size of the core directly influences the particle size of the final function modification magnetic beads, and the function modification magnetic beads with small particle size have higher specific surface area, thereby being more beneficial to DNA adsorption.
Specifically, the silicon dioxide layer is obtained by dehydration condensation of a silane coupling agent, and the thickness of the silicon dioxide layer is 2-8 mu m; alternatively, the silane coupling agent may be one or more of tetramethoxysilane, tetraethoxysilane, tetrapropoxysilane, and tetrabutoxysilane.
Further, the mass ratio of the silane coupling agent to the fluorocarbon is (46-187) to (1.5-3). Alternatively, the mass ratio may be 46:1.5, 187:3, 100:2, 46:3, 187:1.5.
Specifically, the surface of the silicon dioxide layer is grafted with an amino silane-containing coupling agent; alternatively, the aminosilane-containing coupling agent may be one or more of 3-aminopropyl triethoxysilane, gamma-aminopropyl triethoxysilane and 3-aminopropyl trimethoxysilane. Alternatively, if the silica layer is prepared using an amino-containing silane coupling agent, the amino-containing silane coupling agent may be grafted differently on the surface of the silica layer.
Specifically, the mass ratio of the amino-containing silane coupling agent to the dimethyl octenamide dihydrochloride is (46-187) to (5-10). Optionally, the mass ratio is 46:10, 46:5, 187:5, 187:10, 100:8.
The invention also provides a preparation method of the functional modification magnetic bead, which comprises the following steps:
dispersing the magnetic beads in water, and then adding functional groups to perform covalent bonding reaction, namely the functional modified magnetic beads; the magnetic beads are Fe with silicon dioxide layers coated on the surfaces 3 O 4 Particles (which may be micro-scale or nano-scale) with the functional groups being fluorocarbon and/or dimethyl octenamide dihydrochloride.
Specifically, the magnetic beads are prepared by the following preparation method:
fe is added to 3 O 4 Dispersing the particles in an ethanol-glycerol mixed solution, adding tetraethyl silicate and an ammonium chloride solution, and reacting for 12-20 hours at room temperature to obtain magnetic beads;
the volume ratio of ethanol to glycerol in the ethanol-glycerol mixed solution is 1: (0.5-1.5).
Preferably, the volume ratio of ethanol to glycerol in the ethanol-glycerol mixed solution is 1:1.
the application of the functional modified magnetic beads in extraction and/or purification is also within the protection scope of the invention.
The invention also provides a DNA gel recovery kit comprising the functional modification magnetic beads.
Specifically, the DNA gel recovery kit comprises a melting/binding buffer solution, mag-Bind Particles, a washing solution 1, a washing solution 2 and an eluent;
(1) The melting/binding buffer is Sodium Perchloride, HCl, sodium Acetate and H 2 A mixed solution of O; the preparation method comprises the following steps: 6 to 10 of M Sodium Perchloride, 100 to 300mM of HCl, 1 to 2M of Sodium Acetate and H 2 O is in a volume ratio of 7.5:0.125:0.1:2.275, and the pH value of the prepared melting/combining buffer solution at room temperature is 4-5;
(2) The Mag-Bind Particles are the functional modified magnetic beads;
(3) The washing liquid 1 is a high-salt washing liquid, and the preparation method comprises the following steps: uniformly mixing GNC aqueous solution with the volume concentration of 40-60% with absolute ethyl alcohol with the volume of 1.27 times;
(4) The washing liquid 2 is a low-salt high-alcohol washing liquid, and the preparation method comprises the following steps: the mass concentration of the solution is 5 percent of NaCl solution and 10mM Tris-HCl according to the volume ratio of 5 percent: mixing the mixed aqueous solution with 95% of absolute ethyl alcohol with the volume of 2.3 times to obtain the water-based paint;
(5) The eluent is an Elution Buffer.
In practical application, the DNA gel recovery method based on the DNA gel recovery kit comprises the following steps:
(1) Sol: cutting target DNA fragments from agarose gel, transferring the target DNA fragments into a centrifuge tube, weighing the sliced gel, adding 2 times of volume of melting/binding buffer solution, and incubating at 60-70 ℃ for 7-10min to melt the gel;
(2) DNA binding: adding Mag-Bind Particles to the mixed solution obtained in the step (1), and swirling or shaking at 60-70 ℃ for 10-30min to Bind DNA to the Mag-Bind Particles; the centrifuge tube was then placed on a magnetic rack, the Mag-Bind Particles were collected, allowed to stand at room temperature until the Mag-Bind Particles were fully adsorbed to the tube wall, and the supernatant was blotted to give DNA-bound Mag-Bind Particles.
(3) And (3) DNA cleaning: washing the Mag-Bind Particles-DNA mixture obtained in the step (2) with a washing liquid 1 for 1 time; then placing the centrifuge tube on a magnetic rack, and magnetically separating supernatant from Mag-Bind Particles-DNA; washing was repeated 1 time with Mag-Bind Particles-DNA using wash 1; adding a washing solution 2 to wash the Mag-Bind Particles-DNA; the supernatant was magnetically separated from Mag-Bind Particles-DNA to remove proteins and salts.
(4) DNA recovery: any residual liquid in the centrifuge tube on the magnetic rack in the step (3) is sucked by a pipette, and the magnetic rack is placed for 10-20min at room temperature, so that Mag-Bind Particles-DNA is air-dried; taking out the centrifuge tube from the magnetic rack and placing the centrifuge tube on the tube bracket; adding the eluent, and vortexing or shaking for 5-10min to elute DNA bound to Mag-Bind Particles; the centrifuge tube was placed on a magnetic rack, the Mag-Bind Particles were collected, left at room temperature until the Mag-Bind Particles were fully adsorbed to the tube wall, and finally the supernatant containing purified DNA was transferred to a fresh centrifuge tube and stored at-20 ℃.
It should be noted that: the above method can be programmed on an Omega Bio-Tek MagBinder 32 or other similar instrument for automatic extraction.
The invention has the following beneficial effects:
the functional modified magnetic beads of the invention are made of Fe 3 O 4 Core of Fe 3 O 4 The surface of the core is wrapped with a silicon dioxide layer, and the functional modification group is connected through the dioxide layer, so that the average recovery rate of the magnetic beads to DNA is improved by utilizing the interaction of the functional modification group and nucleic acid. The functional modified magnetic beads are applied to a nucleic acid adsorption columnWhen compared with the nucleic acid adsorption column (the recovery rate of nucleic acid is between 50% and 70% and the extraction effect is better for the nucleic acid fragments within the range of 100 bp-20 kb) in the conventional column extraction method, the nucleic acid extraction rate of the functional modified magnetic beads reaches more than 80%, and the nucleic acid adsorption column has a good adsorption effect for the nucleic acid fragments within the range of 50 bp-50 kb.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Drawings
FIG. 1 is a graph showing the particle size distribution of the functionally modified magnetic beads (Mb-FS) of example 1.
FIG. 2 is a graph showing the particle diameter distribution of the functionally modified magnetic beads (Mb-DMS) in example 3.
Example 1
A functionally modified magnetic bead, which can be prepared by the following preparation method:
5g of magnetic beads (Mb) were dispersed in 100mL of deionized water, followed by the addition of 2.5g of Fluorocarbon (FS)
Stirring and combining at 400-600 rpm for 3-5 h at room temperature; and (3) the magnet adsorption product, and washing residual fluorocarbon with deionized water to obtain fluorocarbon-modified magnetic beads (Mb-FS-1).
Wherein, the magnetic beads can be prepared by the following preparation method:
(1) 2.5g of Fe 3 O 4 Stirring (200-500 rpm) the particles (with the particle size of 1 μm) to disperse in ethanol-glycerol mixed solution with the volume ratio of 1:1;
(2) 116g of tetraethyl silicate and 200-400 mL of ammonium chloride solution are respectively added;
(3) Stirring at 800-1500 rpm for reaction for 12-20 h;
(4) Terminating the reaction, adsorbing the product by the magnet, and cleaning with deionized water to obtain the magnetic beads (Mb).
The fluorocarbon-modified magnetic beads (Mb-FS-1) were subjected to particle size distribution measurement, and the average particle size of the fluorocarbon-modified magnetic beads was 6.71. Mu.m, as shown in FIG. 1.
Example 2
A functionally modified magnetic bead, which can be prepared by the following preparation method:
dispersing 5g of magnetic beads (Mb) into 100mL of deionized water, then adding 1.75g of Fluorocarbon (FS), and stirring and combining at 400-600 rpm for 3-5 h at room temperature; the magnet adsorption product is washed off with deionized water to obtain fluorocarbon modified magnetic beads (Mb-FS-2); wherein the magnetic beads are the same as in example 1.
Example 3
A functionally modified magnetic bead, which can be prepared by the following preparation method:
s1, dispersing 5g of magnetic beads (Mb) into 300-600 mL of isopropanol, dropwise adding 7.5mL of (3-Aminopropyl) triethoxysilane (APTES), incubating for 4-8 h by ultrasound, adsorbing a product by a magnet, and washing off residual APTES by deionized water to obtain amino modified silica magnetic beads (Mb-NH) 2 )；
S2, mb-NH 2 Dispersing into 300-600 mL deionized water, adding 9g of dimethyl octenamide Dihydrochloride (DMS), and stirring at 400-800 rpm at room temperature for reaction for 4-8 h;
s3, terminating the reaction, adsorbing the product by using a magnet, and washing off residual DMS by using deionized water to obtain a magnetic bead (Mb-DMS-1) modified by the DMS; wherein the magnetic beads are the same as in example 1.
The above-mentioned amination-modified silica magnetic beads (Mb-NH) 2 ) The particle size distribution was measured, and the measurement result was shown in FIG. 2, and the average particle size of the aminated silica magnetic beads was 6.1. Mu.m.
Example 4
A functionally modified magnetic bead, which can be prepared by the following preparation method:
s1, dispersing 5g of magnetic beads (Mb) into 300-600 mL of isopropanol, dropwise adding 7.5mL of (3-aminopropyl) triethoxysilane ((APTES), carrying out ultrasonic incubation for 4-8 h, removing residual APTES by using magnet adsorption products and deionized water to obtain amino modified silica magnetic beads (M)b-NH 2 )；
S2, mb-NH 2 Dispersing into 300-600 mL deionized water, adding 6g of dimethyl octenamide Dihydrochloride (DMS), and stirring at 400-800 rpm at room temperature for reaction for 4-8 h;
s3, terminating the reaction, adsorbing the product by using a magnet, and washing off residual DMS by using deionized water to obtain a magnetic bead (Mb-DMS-1) modified by the DMS; wherein the magnetic beads are the same as in example 1.
Comparative example 1
A magnetic bead which can be prepared by the following preparation method:
(1) 2.5g of Fe 3 O 4 Stirring (200-500 rpm) the particles (with the particle size of 1 μm) to disperse in ethanol-glycerol mixed solution with the volume ratio of 1:1;
(2) 116g of tetraethyl silicate and 200-400 mL of ammonium chloride solution are respectively added;
(3) Stirring at 800-1500 rpm for reaction for 12-20 h;
(4) Terminating the reaction, adsorbing the product by the magnet, and cleaning with deionized water to obtain the magnetic beads (Mb).
Performance testing
The DNA average recovery rate testing method of the DNA gel recovery kit based on the magnetic bead method comprises the following steps:
(1) Preparing a melting/binding buffer, a diluted washing solution 1 and a washing solution 2:
the preparation method of the melting/binding buffer solution in the step (1) comprises the following steps: 6 to 10 of M Sodium Perchloride, 100 to 300mM of HCl, 1 to 2M of Sodium Acetate and H 2 O is in a volume ratio of 7.5:
0.125:0.1:2.275, and the pH value of the prepared melting/combining buffer solution at room temperature is 4-5;
the washing liquid 1 is a high-salt washing liquid, and the preparation method comprises the following steps: uniformly mixing GNC aqueous solution with the volume concentration of 40-60% with absolute ethyl alcohol with the volume of 1.27 times;
the washing liquid 2 is low-salt high-alcohol washing liquid, and the preparation method comprises the following steps: the mass concentration of the solution is 5 percent of NaCl solution and 10mM Tris-HCl according to the volume ratio of 5 percent: mixing the mixed aqueous solution with 95% of absolute ethyl alcohol with the volume of 2.3 times.
(2) Agarose gel electrophoresis separates the DNA fragments, and when the strips are sufficiently separated, the target DNA fragments are carefully excised with a broad, clean, sharp surgical knife, and excess agarose is removed to reduce the size of the gel sheet.
(3) The sliced gel was weighed in a test tube and after thawing the appropriate volume was determined. The volume of the slice gel thawed can be estimated as follows: 1 gram of gel is equal to 1 milliliter volume.
(4) Based on the estimated gel slice volume, 2 volumes of melting/binding buffer were added. For example, if there is 0.3g of gel, 0.6mL of melting/binding buffer is added.
(5) The test tube is incubated at 60-70 ℃ for 7-10 minutes or until the gel is completely melted. If mixing cannot be continued, vortexing or shaking for 30 seconds every 2-3 minutes.
(6) 5-10 mu L of Mag-Bind Particles are added into a test tube and vortexed or shaken at 60-70 ℃ for 10-30 minutes. If the mixing cannot be continuously carried out, vortexing or shaking for 30 seconds every 2-3 minutes; the Mag-Bind Particles are the functionally modified magnetic beads of examples 1 to 4 and the magnetic beads of comparative example 1, respectively.
(7) The centrifuge tube was placed on a magnetic rack, and the Mag-Bind Particles-DNA was collected and allowed to stand at room temperature until the Mag-Bind Particles-DNA was completely adsorbed to the tube wall.
(8) The supernatant was aspirated. Note that Mag-Bind Particles-DNA is not absorbed.
(9) The centrifuge tube is taken out from the magnetic rack and placed on the tube bracket.
(10) 400-800. Mu.L of washing liquid 1 was added to each test tube and vortexed for 1-2 minutes.
(11) The centrifuge tube was placed on a magnetic rack, and the Mag-Bind Particles-DNA was collected and allowed to stand at room temperature until the Mag-Bind Particles-DNA was completely adsorbed to the tube wall.
(12) The supernatant was aspirated. Note that Mag-Bind Particles-DNA is not absorbed.
(13) The centrifuge tube is taken out from the magnetic rack and placed on the tube bracket.
(14) 400-800. Mu.L of washing liquid 1 was again added to each tube and vortexed for 1-2 minutes.
(15) The centrifuge tube was placed on a magnetic rack, and the Mag-Bind Particles-DNA was collected and allowed to stand at room temperature until the Mag-Bind Particles-DNA was completely adsorbed to the tube wall.
(16) The supernatant was aspirated. Note that Mag-Bind Particles-DNA is not absorbed.
(17) The centrifuge tube is taken out from the magnetic rack and placed on the tube bracket.
(18) 400-800. Mu.L of washing liquid 2 was added to each tube and vortexed for 1-2 minutes.
(19) The centrifuge tube was placed on a magnetic rack, and the Mag-Bind Particles-DNA was collected and allowed to stand at room temperature until the Mag-Bind Particles-DNA was completely adsorbed to the tube wall.
(20) The supernatant was aspirated. Note that Mag-Bind Particles-DNA is not absorbed.
(21) Residual liquid in the magnetic bead tube on the magnetic rack is sucked by a pipette, the magnetic bead tube is placed for 10 to 20 minutes, and the Mag-Bind Particles-DNA is dried.
(22) The centrifuge tube is taken out from the magnetic rack and placed on the tube bracket.
(23) 30 to 50. Mu.L of the eluent was added to each tube, and the mixture was vortexed or shaken at 60 to 70℃for 5 to 10 minutes to elute the DNA bound to the magnetic beads. If stirring cannot be continued, it is vortexed or shaken for 30 seconds every 1 minute for 5 to 10 minutes.
(24) The centrifuge tube was placed on a magnetic rack and the Mag-Bind Particles were collected and allowed to stand at room temperature until the Mag-Bind Particles were fully adsorbed to the tube wall.
(25) The supernatant containing purified DNA was transferred to a new centrifuge tube, and the recovery and purity of DNA were checked, and the remaining sample was stored at-20 ℃.
(26) The DNA products were detected using both the NanoDrop and Pico Green methods, and the results are shown in Table 1. Wherein mean recovery of DAN = mean DNA concentration per group x DNA recovery volume/initial DNA addition x 100%.
TABLE 1 average recovery of DNA from functionally modified magnetic beads in examples 1 to 4 and unmodified magnetic beads in comparative example 1
As can be seen from the data in table 1, the use of fluorocarbon or dimethyl octenamide dihydrochloride modified beads significantly improved the average DNA recovery compared to beads not modified with functional groups.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. A functional modification magnetic bead is characterized in that the functional modification magnetic bead comprises Fe 3 O 4 Core and coating on Fe 3 O 4 A silicon dioxide layer and a functional group modified on the surface of the dioxide layer on the surface of the core;
the functional group is fluorocarbon and/or dimethyl octenamide dihydrochloride; the average particle diameter of the functional modified magnetic beads is 2-10 mu m.
2. The functionally modified magnetic bead of claim 1, wherein the Fe 3 O 4 The average particle diameter of the core is less than or equal to 2 mu m.
3. The functionally modified magnetic bead according to claim 1, wherein the silica layer is obtained by dehydration condensation of a silane coupling agent.
4. The functionally modified magnetic bead according to claim 3, wherein the silica layer has a thickness of 2 to 8. Mu.m.
5. The functionally modified magnetic bead according to claim 3, wherein the mass ratio of the silane coupling agent to the fluorocarbon is (46 to 187) to (1.5 to 3).
6. The functionally modified magnetic bead according to claim 1, wherein the silica layer surface is grafted with an aminosilane-containing coupling agent;
the mass ratio of the amino-containing silane coupling agent to the dimethyl octenamide dihydrochloride is (46-187) to (5-10).
7. A method of preparing the functionally modified magnetic bead according to any one of claims 1 to 6, comprising the steps of:
dispersing the magnetic beads in water, then adding functional groups, and carrying out covalent bonding reaction to obtain the functional modified magnetic beads;
the magnetic beads are Fe with silicon dioxide layers coated on the surfaces 3 O 4 Particles, wherein the functional group is fluorocarbon and/or dimethyl octenamide dihydrochloride.
8. The method of claim 7, wherein the magnetic beads are prepared by the method of:
fe is added to 3 O 4 Dispersing the particles in an ethanol-glycerol mixed solution, adding tetraethyl silicate and an ammonium chloride solution, and reacting for 12-20 hours at room temperature to obtain magnetic beads; the volume ratio of the ethanol to the glycerol in the ethanol-glycerol mixed solution is 1 (0.5-1.5).
9. Use of the functionally modified magnetic beads according to any one of claims 1 to 6 for extraction and/or purification.
10. A DNA gel recovery kit comprising the functionally modified magnetic beads of any one of claims 1 to 6.
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